Influenza sensor

ABSTRACT

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

BENEFIT OF PRIOR APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Application No. 60/162,427, filed Oct. 28, 1999

RELATED APPLICATION

This application is a divisional of Ser. No. 09/699,225, filed Oct. 27,2000 now U.S. Pat. No. 6,627,396, by Swanson et al.

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the U.S. Department of Energy. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a diagnostic sensor for the detectionof influenza virus and to a method of detecting influenza virus withsuch a diagnostic sensor.

BACKGROUND OF THE INVENTION

The early diagnosis of influenza infection is important for severalreasons. One reason is that it is critical to be able to rapidly screeninfluenza from other infectious diseases in the event of a bio-agentattack. Most scenarios for bio-agent attacks show a slowed response tothe recognition that an attack has taken place primarily becausediseases such as anthrax and smallpox present flu-like symptoms. Medicalpersonnel do not have a rapid and simple screen for influenza infectionand, consequently, victims can be miss-diagnosed as having the flu andsent home. A delay of even a few days in the recognition of a bio-agentattack can have adverse affect on the minimization of the impact of anattack.

Another reason for a rapid diagnostic for influenza is important is inhelping to avert a worldwide pandemic in the event that a new strainlike the 1918 swine flu appears. Rapid screening with inexpensivefieldable sensors is essential to rapidly pinpoint a new potentialoutbreak. Although it is also important to specify the strain of theinfluenza infection, it is first critical to rapidly identify anoutbreak and this can only be done using a flexible, inexpensive,fieldable sensor.

Recently, a number of high binding affinity neuraminidase (also known assialidase) inhibitors have been developed and shown to be quiteeffective in curing the flu but only if such inhibitors are administeredearly on in the infection (generally within the first 24 to 48 hours).At present, these drugs can not be effectively used as there is not asimple diagnostic tool that can be used to detect the influenza virusearly enough to effectively use neuraminidase inhibitors. The onlytechnologies currently capable of early diagnosis of influenza arelab-based approaches like ELISA, which are instrument and personnelintensive, expensive, and slow. What is needed is a simple inexpensivediagnosis that can be easily used in either a clinical or field settingand yet have at least the same specificity and sensitivity as ELISA.Accordingly, it is highly desirable to develop a rapid diagnosis forinfluenza to facilitate the treatment of influenza using suchneuraminidase inhibitors.

An optical biosensor system has recently been developed to rapidlydetect protein toxins, e.g., cholera, shiga, and ricin (see, U.S. patentapplication Ser. No. 09/338,457, by Song et al., filed Jun. 22, 1999).The integrated optical biosensor developed for the detection of proteintoxins was based on proximity-based fluorescence changes that aretriggered by protein-receptor binding. In demonstrations of thisapproach for the detection of cholera and avidin using flow cytometry,it was shown that this technique was as sensitive as ELISA. In contrastto ELISA, such an optical biosensor can be much faster (minutes),simpler (a single step with no added reagents) and robust owing to thestability of the recognition molecules (glycolipids and biotin) andmembranes. More recently, an optical biosensor system has beenincorporated into planar optical waveguides (see, U.S. ProvisionalPatent Application Ser. No. 60/140,718, by Kelly et al., filed Jun. 22,1999) and shown to have sensitivity equivalent to that of flowcytometry. The demonstration of such an optical biosensor using planaroptical waveguides provides a path towards the development ofminiaturized sensor arrays.

One object of the present invention is adaptation of such a biosensor tosensing applications directed to the detection of influenza virus.

Another object of the present invention is the selection and chemicalmodification of receptors that bind neuraminidase and that allowattachment of such receptors to membranes together with theincorporation of fluorophores.

SUMMARY OF THE INVENTION

To achieve the foregoing and other objects, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the present invention provides for the detection of tetramericneuraminidase within a sample, where a positive detection indicates thepresence of a target virus within said sample, said sensor including asurface, recognition molecules situated movably at said surface, saidrecognition molecules capable of binding with said tetramericmultivalent neuraminidase, said recognition molecules furthercharacterized as including a fluorescence label thereon, and, a meansfor measuring a change in fluorescent properties in response to bindingbetween multiple recognition molecules and said tetramericneuraminidase.

The present invention further provides a method of method of detectingtetrameric neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said methodincluding contacting a sample with a sensor including a surface,recognition molecules situated movably upon said surface, saidrecognition molecules capable of binding with said tetramericmultivalent neuraminidase wherein said recognition molecules include afluorescence label thereon, and measuring a change in fluorescentproperties in response to binding between multiple recognition moleculesand said tetrameric neuraminidase.

The present invention further provides for the detection of tetramericneuraminidase within a sample, where a positive detection indicates thepresence of a target virus within said sample, said sensor including asurface, at least two different recognition molecules situated movablyupon said surface, said recognition molecules capable of binding withsaid tetrameric multivalent neuraminidase wherein at least onerecognition molecule includes a fluorescence donor label thereon and atleast one recognition molecule includes a fluorescence acceptor labelthereon, and, a means for measuring a change in fluorescent propertiesin response to binding between at least two different multiplerecognition molecules and said tetrameric neuraminidase.

The present invention further provides a method of method of detectingtetrameric neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said methodincluding contacting a sample with a sensor including a surface, atleast two different recognition molecules situated movably upon saidsurface, said recognition molecules capable of binding with saidtetrameric multivalent neuraminidase wherein at least one recognitionmolecule includes a fluorescence donor label thereon and at least onerecognition molecule includes a fluorescence acceptor label thereon, andmeasuring a change in fluorescent properties in response to bindingbetween multiple recognition molecules and said tetramericneuraminidase.

The present invention still further provides a trifunctional compositionof matter including a trifunctional linker moiety including as groupsbonded thereto (a) an alkyl chain adapted for attachment to a substrate,(b) a fluorescent moiety capable of generating a fluorescent signal, and(c) a recognition moiety having a spacer group of a defined lengththereon, said recognition moiety capable of binding with tetramericmultivalent neuraminidase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of an exemplary receptor molecule including thethree necessary functionalites.

FIG. 2 shows a diagram of another exemplary receptor molecule includingthe three necessary functionalites.

DETAILED DESCRIPTION

The present invention concerns a diagnostic sensor for the detection ofinfluenza virus and to a method of detecting influenza virus with such adiagnostic sensor. In particular, the present invention concerns adiagnostic sensor capable of detecting organisms such as influenza virusthat contain neuraminidase.

Organisms that contain neuraminidase include bacteria (Vibrio cholerae,Clostridium perfringens, Streptococcus pneumoniae, and Arthrobactersialophilus) and viruses (influenza virus, parainfluenza virus, mumpsvirus, Newcastle disease virus, fowl plague virus, and sendai virus). Inviruses, neuraminidase occurs as a tetramer. This tetrameric structurefacilitates the operation of the sensor of the present invention.Detection of neuraminidase activity related to any tetramericneuraminidase structure in a virus is within the scope of the presentinvention. Detection of neuraminidase from influenza virus isparticularly desired.

The selection of receptors is related to the prior work that has beendone to synthesize neuraminidase inhibitors having high bindingaffinities. In principal, any neuraminidase inhibitor could be used butpreferably the present influenza sensor will incorporate thoseneuraminidase inhibitors that have the highest binding affinities.Typically, neuraminidase inhibitor compositions having in vitro K_(i)(inhibitory constants) of less than about 5×10⁻⁶M, typically less thanabout 5×10⁻⁷M and preferably less than about 5×10⁻⁸M are excellentcandidates for the recognition portion of the influenza sensor of thepresent invention.

In one embodiment, the influenza sensor of the present inventioninvolves: 1) formation of a biomimetic membrane, which incorporatesfluorescent dye-labeled receptors for neuraminidase, on the surface ofan optical transducer (this could be a glass bead for flowcytometry—FCM—or a planar optical waveguide for an integrated opticalbiosensor); 2) the chemical modification of selected neuraminidaseinhibitors to attach them to a membrane and to also attach fluorophoresthat fold into the fluid upper leaf of the membrane; 3) the detection,using a planar optical waveguide of a microsensor array or FCM, ofenvelope proteins or the influenza viral particle directly as measuredby a shift in the ratios of intensity of two individualfluorescently-emitted signals, and 4) the use of multiple receptors inthe recognition effect (in influenza neuraminidase is a tetramer) toinsure extremely high effective binding affinities (avidity) and, as aresult, ultrahigh sensitivities and specificities.

One advantage of the present invention is the ultrahigh sensitivity andspecificity obtained by using multiple receptors each with high bindingaffinities (the avidity effect) to help insure early diagnosis, e.g.,within the first 12 hours after infection. Another advantage of thepresent invention is the simplicity and speed of operation which makesdetection fast and operation possible in a variety of situations.Another advantage of the present invention is the elimination of theneed for additional reagents or additives thereby simplifying the useand extending shelf storage lifetimes. Another advantage of the presentinvention is the flexibility in adaptation to either flow cytometry orto miniaturized sensor systems utilizing planar optical waveguidepermits use in a variety of clinical or field situations. Anotheradvantage of the present invention is the robustness of the sensorsystem that results from the high stability of the receptor moleculesand the active membrane. Another advantage of the present invention isthe simplicity of sample introduction which minimizes front-end samplepreparation.

Coupling recognition to signal transduction and amplification can beconducted as follows. The sensor of the present invention mimics manycell signaling processes in nature by directly coupling a recognitionevent to signal transduction and amplification. In the case of thepresent influenza sensor, the sensor relies on recognition of chemicallymodified sialic acid-like receptors by neuraminidase, an envelopeprotein for influenza. As neuraminidase in a virus such as influenza isa multivalent protein (neuraminidase is a tetramer), binding will bringseveral receptors into close proximity thereby triggering proximitybased fluorescence changes. The selection of target receptors isdiscussed below. The receptor molecules are chemically modified to bothattach fluorescent tags and to attach them to the fluid upper leaf of aphospholipid bilayer. The choice of a phospholipid bilayer is importantfor several reasons. First, this is an excellent mimic of a cellmembrane surface, which is the natural target of envelope viruses.Second, the use of membrane mimics helps minimize non-specific proteinabsorption and the attendant nonspecific response of the sensor element.Third, the upper leaf of the membrane is fluid thereby insuring that thereceptor molecules and their fluorescent tags are mobile and, if theconcentration is low, relatively distant thereby minimizing proximitybased fluorescence changes prior to protein binding. The binding eventbetween neuraminidase and the sialic acid-like receptors then bringsmultiple receptors into close proximity triggering the fluorescencechanges. There are two proximity based fluorescence changes that can beused for detection. The simplest is self-quenching of fluorescence thatresults in a sharp decrease in the fluorescence of the fluorophoreattached to the receptor molecules. The second is resonant energytransfer (FRET) where donor fluorophores transfer their energy to theacceptor fluorophores that are attached to the receptor molecules. Inthis case, the receptor molecules are tagged with both fluorescentdonors and acceptors (typically in a 1:1 ratio). FRET results in a colorchange in the fluorescence, which can be more easily distinguished fromsimple self-quenching in terms of being directly coupled to thereceptor-protein recognition. Preferably, the selection of fluorophoresis such that there must be overlap of the donor emission with theexcitation profile of the acceptor. Many pairs of fluorophores exhibitthis relationship and the selection is primarily dictated by thestability of the fluorophores and the most effective separation of theemission profiles to minimize background fluorescence.

Influenza is an RNA virus and, therefore, has a rapid antigenic driftand antigenic shift. As a result, the binding of antibodies and receptormolecules to neuraminidase is constantly changing even within aparticular strain. However, antigenic shift and drift do not affectbinding of a neuraminidase inhibitor which bind to silacic acid. Forthis reason, the present invention has targeted the binding region ofneuraminidase that targets sialic acid residues on the cell membranesurface. It is through neuraminidase binding to sialic acid that theinfluenza virus particle invades the host cell through membrane fusion.As this binding event is critical to viral particle invasion, it islikely that the binding pocket in neuraminidase that selects sialic acidis relatively invariant. There have been several crystal structures ofneuraminidase measured with differing inhibitors (molecules that mimicsialic acid but which have even higher binding affinities toneuraminidase) that show the binding pocket for sialic acid site isrelatively invariant. Moreover, there has been a great deal of work insynthesizing neuraminidase inhibitors that have exceptionally highbinding affinities and these molecules are all good potential receptorsfor the influenza sensor of the present invention. As noted below,initial selection was of a few molecules that have binding affinities inthe micromolar range. The use of molecules that target the sialic acidbinding site insures that this sensor will be effective over a longperiod of time and to virtually any strain of influenza. Moreover, asneuraminidase is multivalent with regard to binding sialic acid, the useof inhibitors and sialic acid variants insures that the above sensortransduction scheme (proximity based fluorescence changes) can work and,equally important, that the effective binding affinities are high byvirtue of the avidity effect.

Chemical modification of the neuraminidase receptors can be as follows.A number of neuraminidase inhibitors derived from 3,4-diamino benzoicacid have been described in the literature. These compounds bind to thesialic acid binding site on neuraminidase. One of these 3,4-diaminobenzoic acid-based neuraminidase inhibitors, 4-acetylamino-3-guanidinebenzoic acid is reported to bind NA with an affinity constant of 10⁵.The present approach to a neuraminidase detector involves covalentlylinking this inhibitor to a fluorescent molecule that is anchored into amembrane. The covalent attachment is accomplished as follows.3,4-Diamino benzoic acid (I) is acetylated on the 4-amino group to yield3-amino-4-acetylamino benzoic acid (II). Compound II is then alkylatedto yield 3-alkylamino-4-acetylamino benzoic acid (III). Treatment of IIIwith cyanogen bromide followed by ammonia yield compound IV that mayserve as a neuraminidase receptor in the present invention.

The neuraminidase receptor (IV) can be linked to the fluorescent probe anumber of ways. One possible approach is as follows. First compound IVis attached to a polyethylene glycol spacer to the alpha-amino group oflysine. The alpha-carboxyate is modified as an amide to a 16, 17, or18-carbon hydrocarbon, which serves as a membrane anchor. The ε-aminogroup of lysine is modified to carry a hydrophobic fluorescent moleculesuch as a Bodipy molecule.

Preparation of the Membrane Architectures on Optical Transducers can beas follows. As noted above, the receptor molecules would have beenchemically modified to bind them into the upper leaf of a phopholipidbilayer through attachment to two aliphatic side chains. Moreover, thefluorophore has been selected and attached in a way that insures that itfolds over into the upper leaf of the bilayer. The fact that thefluorophore resides in the upper leaf of the bilayer is important fortwo reasons. First, this insures that the fluorophore itself and thelinker that attaches it to the lipid tail do not interfere withrecognition by providing a non-specific site for protein binding.Second, the residence of the fluorophore in the upper leaf providesadditional stability of the receptor-membrane structure. The membranesare then fabricated onto optical transducers (either glass beads for FCMor planar optical waveguides for microsensor arrays) by vesicle fusiononto hydrophobic or hydrophilic surfaces. The simplest approach isvesicle fusion onto a hydrophilic surface to form a support bilayer.This can be done in a flow cell where the surface can be exposed for aperiod of time (hours) to a solution containing the vesicles. The secondapproach is to spread vesicles containing the tagged receptors over amethyl terminated self-assembled monolayer to form a hybrid bilayerwhere the lower leaf is covalently attached to the transducer surface.The hybrid bilayer has the advantage of better long-term stability.

In the present invention, the architecture of the fluid membranes can beas a regular bilayer membrane where both layers are deposited upon asupport surface, can be a hybrid bilayer, e.g., where a first layer iscovalently attached to an oxide surface, can be a selectively tetheredbilayer on an oxide surface, where a membrane molecule is covalentlybonded to the oxide substrate, or a bilayer cushioned by a polymer film.Supported membranes useful in the practice of the present invention aregenerally described by Sackmann, in “Supported Membranes: Scientific andPractical Applications”, Science, vol. 271, no. 5245, pp. 43-45, Jan. 5,1996. Hybrid bilayer membranes or selectively tethered bilayer membranesmay be more preferred as such membranes may have greater stability overtime and therefor provide greater shelf lifetimes for sensorapplications. Additionally, a surface with mobile receptors such as anoxide surface with mobile receptor molecules thereon can serve as aplatform in the present invention.

Bilayer membranes can be formed upon a planar oxide substrate, e.g., byinitially forming vesicles followed by vesicle fusion or spreading of,e.g., phospholipid, bilayers on glass substrates as is well known tothose skilled in the art.

In one embodiment of the present invention, the transduction elementused is fluorescein, which has a high extinction coefficient, a highfluorescence quantum yield and proximity-dependent fluorescenceself-quenching. Other suitable fluorescent dyes are well known to thoseof skill in the art. Fluorescein may be covalently attached to a freefunctional group by appropriate coupling to produce afluorescein-labeled moiety. The fluorescein should have minimalinfluence on the binding affinity of the recognition portion of thefinal molecule to the influenza virus.

In another embodiment of the present influenza sensor, the sensingmolecules can be functionalized with either an acceptor dye molecule ora donor dye molecule whose excitation spectra overlap for efficientenergy transfer. In effect, excitation of a blue emitting dye can resultin fluorescence with a maximum at roughly 570 nm when functionalizedBodipy is free to move about in the bilayer membrane. Upon exposure toinfluenza virus (tetrameric neuraminidase), both the donor and theacceptor dyes are brought into close proximity. This can result in anenergy transfer and a decrease in the fluorescence at 570 nm and aconcomitant increase in the fluorescence of the acceptor dye that hasits fluorescence maximum at roughly 630 nm. Such a simultaneous increasein the red fluorescence and decrease in the blue fluorescence is ahighly distinguishing feature of this sensor approach. In effect, atwo-color fluorescence measurement can be used to probe the intensity offluorescence from both dye molecules. Only a specific binding eventbetween the neuraminidase and the receptor will give rise to such asimultaneous increase in one fluorescent signal with a decrease in theother. Any change in the environment will give rise to shifts of thefluorescence of both dye molecules. Such an energy transfer approachprovides a means for self-referencing in such sensor applications.

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, workup procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Workup typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reactionby-products and use of anhydrous reaction conditions (e.g. inert gasenvironments) are common in the art and will be applied when applicable.

As discussed in this application, in a preferred embodiment the signaltransduction scheme is dependent of FRET. To be detected, influenzavirus particles (neuraminidase tetramers) must bind two or more receptormolecules. Binding must cause the receptors to aggregate resulting influorescence energy transfer. These “receptor molecules” have threefunctions. First they must have a recognition ligand that bindsspecifically to an agent. The receptor must carry the fluorescentreporter and must be mobile in a lipid bilayer membrane. Diagrammed inFIG. 1 is a prototype “receptor”. A trifunctional linker molecule mustconnect the recognition site, fluorescent reporter, and membrane anchor.

Trifunctional Linker—Because they are available, α-amino acids withfunctional groups on the side chains are good candidates astrifunctional linkers. In addition to the α-amino and α-carboxylfunctional groups, common amino acids are available with hydroxyl(serine), carboxyl (glutamate and aspartate), thiol (cysteine) and amino(lysine) functionality in the side chain. Lysine derivatives areavailable with the α-amino and ε-amino groups differentially blocked sotheir chemistry is orthoganal. Influenza receptors are prepared fromcommercial N^(α)-benzyloxycarbonyl-N^(ε)-t-butyloxycarbonyl-L-lysineN-hydroxysuccinimide ester (1, Z-Lys(Boc)-Osu). The N^(α)- andN^(ε)-blocking groups are removed differentially by hydrogenation (CBZ)or dilute acid hydrolysis (Boc). The alkyl anchors are added first bydisplacement of the N-hydroxy-succinimide ester by treatment ofZ-Lys(Boc)-Osu with distearylamine (2, Scheme 1). Next the CBZ group isremoved by hydrogenation under standard conditions. Dialkyl-substitutedamide (4) have been prepared in essentially quantitative yield as thestarting material for the synthesis of the artificial influenzareceptors described here. Recognition ligand is attached through aspacer to the α-amino group. Next the Boc protecting group is removedand the fluorescent probe attached to the ε-amino group.

The neuraminidase in influenza virus is uniquely a homotetramericprotein. An active site-specific binder of neuraminidase would provide aflu-specific detector using the FRET scheme. Each viral particlecontains several hundred copies of the neuraminidase tetramer. Inaddition, many competitive inhibitors of flu neuraminidase have beendeveloped as anti-viral agents. These inhibitors provide a specificprobe for the active site of neuraminidase. One example,4-acetylamino-3-guanidino benzoic acid (11) binds flu neuraminidase witha 10 micromolar (μm) affinity constant (Sudbeck et. al., Journal ofMolecular Biology, vol. 267, pp. 584-594 (1997)). Derivatives of thesecompounds have been prepared that will be linked to the receptor througheither 4-amino function or the 3-guanidino group. Other nmolarinhibitors are also becoming available from the pharmaceutical industry.

Among the numerous neuraminidase inhibitors taught by the prior art arethoose compounds described by Luo et al., in U.S. Pat. No. 5,453,533, byBischofberger et al., in U.S. Pat. No. 5,763,483, by Bischofberger etal., in U.S. Pat. No. 5,952,375, Bischofberger et al., in U.S. Pat. No.5,958,973, Kim et al., in U.S. Pat. No. 5,512,596, Kent et al., in U.S.Pat. No. 5,886,213, Babu et al., in U.S. Pat. No. 5,602,277, by vonIzstein et al., in U.S. Pat. No. 5,360,817, Lew et al., in U.S. Pat. No.5,866,601, by Babu et al., in WO97/47194A1, by Babu et al., inWO99/33781A1, and by Brouillette et al., in WO99/14191A1. Each of thesevarious neuraminidase inhibitors may be structurally incorporated intothe influenza sensor of the present invention. The various neuraminidaseinhibitors taught by these enumerated patents are incorporated herein byreference.

Many of the neuraminidase inhibitors having the highest bindingaffinities include at least one functionality from among carboxylate,guanidinium and N-acetyl groups.

Scheme 2 shows the synthesis of the neuraminidase ligand linked via theguanidino group. Commercially available 4-amino benzoic acid (5) isconverted to its methyl ester (6) by treatment with methanol/HCl. Such amethylation is described by Haslam, Tetrahedron, 1980, 36, 2409. Methyl4-amino benzoic acid (6) is treated with acetic anhydride to yieldmethyl 4-acetylamino benzoic acid (7). Such a treatment is described byGreene, T. W., “Protective Groups in Organic Synthesis” (John Wiley &Sons, New York, 1981), in particular at pages 251-252. Treatment of (7)with one equivalent of nitronium tetrafluoroborate in methylene chlorideyields methyl 3-nitro-4-acetylamino benzoic acid (8). Such a treatmentis described by Ottoni et al., Tetrahedron Lett. (1999), 40(6), 1117.Hydrogenation of (8) over Pd/C in ethanol yields the methyl3-amino-4-acetylamino benzoic acid (9). Such a hydrogenation isdescribed by Entwistle et al., J. Chem. Soc., Perkin Trans 1, 1977, 433.Product 9 is alkylated with one equivalent of a spacer molecule where nis generally from about 2 to about 6. Such a alklyation is described byOnaka et al., Chem. Lett., 1982, 11, 1783. The secondary amine is thenconverted to the guanidino function by treatment with cyanogen bromidefollowed by ammonia yielding compound 11. Such a conversion is describedby Rai et al., Indian J. Chem., Sect. B, 1976, 14B(5), 376; by Pankratovet al., Izv. Akad. Nauk SSSR, Ser. Khim. 1975, 10, 2198; and by March,“Advanced Organic Chemistry, 4th edition”, (John Wiley & Sons, New York,1992), in particular at page 903. A fluorescent group is then added tothis ligand using the steps below.

The recognition ligand (11) is then attached via the spacer to the lipidanchor as outlined in Scheme 3. The free carboxylate on the spacer ofcompound 11 is attached to the α-amino group of the lysine linker (4)with dicyclohexyl carbodiimide using standard peptide synthesisconditions to yield (13). Such a process is described by March,“Advanced Organic Chemistry, 4th edition”, (John Wiley & Sons, New York,1992), in particular at page 420. The methyl ester is saponified from 13by treatment with lithium hydroxide in THF/water to yield (14). Such aprocess is described by March, “Advanced Organic Chemistry, 4thedition”, (John Wiley & Sons, New York, 1992), in particular at page383. Facile removal of the Boc group from (14) with trifluoroacetic acidis required before introduction of the fluorescent group. Such a processis described by March, “Advanced Organic Chemistry, 4th edition”, (JohnWiley & Sons, New York, 1992), in particular at page 168. The BODIPYfluorophors are available as their N-hydroxy-succinimide esters.Displacement of the O-Su ester with the ε-amino group of the lysinelinker will yield the final influenza receptor (Scheme 4).

Scheme 4 shows the synthesis of a neuraminidase ligand linked via the4-amino group of the 4-acetylamino-3-guanidino benzoic acid-basedinhibitors. Methyl 4-amino benzoic acid (6) is used as the startingmaterial. Treatment of 6 with the acyl chloride 16 yields the amide 17.The 3-amino function is added by treatment of 17 with nitroniumtetrafluoroborate followed by hydrogenation as described in scheme 2.Conversion of the amino function into a guanidino group is accomplishedby treatment with cyanogen bromide followed by ammonia. The terminalhydroxyl on the spacer of compound 20 must be oxidized to a carboxylatefor attachment to the lysine linker. The free carboxylate onamino-linked neuraminidase ligand (21) is attached to the α-amino groupof the lysine linker (4) with dicyclohexyl carbodiimide as described inscheme 3.

Within the context of the invention samples suspected of containingneuraminidase include natural or man-made materials such as livingorganisms; tissue or cell cultures; biological samples such asbiological material samples (blood, serum, urine, cerebrospinal fluid,tears, sputum, saliva, tissue samples, and the like); laboratorysamples; food, water, or air samples; bioproduct samples such asextracts of cells, particularly recombinant cells synthesizing a desiredglycoprotein; and the like. Typically the sample will be suspected ofcontaining an organism which produces neuraminidase, frequently apathogenic organism such as a virus. Samples can be contained in anymedium including water and organic solvent/water mixtures. Samplesinclude living organisms such as humans, and man made materials such ascell cultures.

In another embodiment, a varied synthesis of the generic linker moleculecan be conducted. As discussed above the signal transduction scheme isdependent on FRET induced by aggregation of two or more fluorescentlytagged antibodies bound to a common surface. These “receptor molecules”have three functions. First, they must have a recognition ligand thatbinds specifically to an agent. For detection of influenza, therecognition ligands can be any neuraminidase inhibitors. In addition,the receptor must carry the fluorescent reporter and must be mobile in alipid bilayer membrane. Diagrammed in FIG. 2 is a prototype “receptor”.Synthetic schemes to prepare the this receptor are diagrammed in schemes5-6. A trifunctional linker molecule, homoserine connects therecognition ligand, fluorescent reporter, and membrane anchor. Based onresults obtained in developing a cholera sensor, this prototype receptorhas the following design characteristics. It has two C-18 alkyl chainsneeded to tightly anchor the receptor in the membrane. The fluorescentreporters are BODIPY-dyes, which are hydrophobic and tethered to thereceptor so as to allow the dye to insert into the lipid bilayer.Insertion into the fluid upper leaf of the bilayer shields the dyemolecule from non-specific protein-dye interactions, provides long termstability towards hydrolysis and helps to anchor the antibody to themembrane. The phosphoryl (PEG)_(n)-spacer will partition into theaqueous phase and has been extensively studied for use as in preparingbio-compatible surfaces. PEG is known to minimize non-specificprotein-surface interactions. (see literature references 20, 23, 27, 41,and 55-57) The length of the spacer can be adjusted by adding more PEGmonomers to optimize fluorescent energy transfer and binding.

The synthesis of the prototype receptor is outlined in detail in schemes5 and 6. For each step literature references are included and the listof references is below. In addition, yields are included for some steps.Homeserine was chosen as the trifunctional linker because is not subjectto elimination as is serine. Two routes are being explored to link thespacer to homoserine through either a phospodiester or a sulfone. Bothof the phosphate and sulfone groups are expected to partition into theaqueous phase. While the phosphodiester linkage is more similar tophospholipids, the potential advantage of the sulfone is its stabilityto hydrolysis. A common intermediate in both routes is compound IV.Commercially available, N-Fmoc O-Trityl homoserine (I) was coupled todioctadecylamine (II) using standard peptide coupling conditions toincorporate the membrane anchors (FIG. 2). (see literature references 3,12, 29, and 53) Removal of the trityl-protecting group withtrifluoroacetic acid frees the hydroxymethyl group of homoserine (IV)for addition of the spacer. (4-6) In the phosphodiester route, treatmentof homeserine (IV) with (tBuO)2P(N(iPr)2) in the presence of

tetrazole follow by deblocking with trifluoroacetic acid gave thephospho homoserine derivative (V) in an overall yield of 73%. (seeliterature references 33, 37-39, 46, 50, 51, and 54) Standardphosphonate DNA synthesis conditions were used for the condensation ofthe PEG spacer (VI) with the phospo homoserine (V). (see literaturereferences 9, 10, and 40) Oxidation with t-butyl hodroperoxide yieldedthe phosphodiester (VII). (see literature references 2, 17, 21, and 43)The intermediate VII has been prepared, purified and characterized byNMR spectroscopy (overall yield 60%). In the sulfone route, conversionthe hydroxyl group of homoserine to its corresponding bromide (Va) wasachieved in 73% yield by treatment with triphenyl phosphine and carbontetrabromide. (see literature references 26, 28, and 49) Nucleophilicsubstitution of bromide by a thiol-terminated PEG spacer (VIa) (18, 48)followed by oxidation will give the sulfone (VIIa). (see literaturereferences 19, 22, 30, 44, and 52)

To complete the linker, the terminal amino group on the PEG spacer isfreed, a thiol carboxy, amino, or aldehyde reactive group is added andthen the BODIPY dye is added. As diagrammed in scheme 5, this scheme isdepicted only for the phopodiester-linked receptor. The identical schemewill be carried out on VIIa to finish the sulfone-linked receptor.First, the BOC-amino protecting group is removed from VII under acidicconditions. (see literature references 4-6) Next reactive group forspecific linkage to recognition ligands is added to the PEG-terminalamino group. For example, a thiol specific disulfide(see literaturereference 11) or maleimide derivative(see literature reference 32) isadded to react with a free thiol on the neuraminidase inhibitor.Similarly, an aldehyde specific hydrazone is added to react with thereducing terminal sugar on a sialaic acid containing oligosaccharide.(see literature references 16, 31, 35, and 36) These reagents,

diagrammed in FIG. 4, are available as activated N-hydroxysuccinimideesters (Pierce Chemical Co.) (see literature references 1, 7, 45, and47), which will react directly with the free amino group to form anamide linkage. (see literature references 8, 25, and 34) The BODIPY dyeis added in “one pot step” involving removal of the Fmoc group from thehomoserine amino group,(see literature references 13-15) which will bemodified with the N-hydroxsuccinimide esters of one of the BODIPY dyes.(see literature references 24 and 42) DCC is dicyclohexylcarbodiimide.HOBT is 1-hydroxybenzotriazole. DEAD is diethyl azodicarboxylate. TFA istrifluoroacetic acid.

Literature Cited

-   1. Ali, M. S., and S. M. Quadri. 1996. Maleimido Derivatives of    Diethylenetriaminepentaacetic acid and    Triethylenetetra-aminehexaacetic Acid: Their synthesis and potential    for specific conjugation with biomolecules. Bioconjugate Chem.    9:645-654.-   2. Andrews, D. M., J. Kitchin, and P. W. Seale. 1991. Solid-phase    synthesis of a range of O-phosphorylated peptides by post-assembly    phosphitylation and oxidation. Int. J. Pept. Protein Res.    38(5):469-475.-   3. Atherton, E., and R. C. Sheppard. 1989. Solid phase peptide    synthesis—a practical approach. IRL Press, Oxford.-   4. Barlos, K., D. Gatos, O. Chatzi, S. Koutsogianni, and W.    Schaefer. 1993. Solid phase synthesis using trityl type side chain    protecting group. Pept. 1992, Proc. Eur. Pept. Symp. 22nd:283-284.-   5. Barlos, K., D. Gatos, S. Koutsogianni, W. Schafer, G.    Stavropoulos, and Y. Wenqing. 1991. Preparation and application of    N-Fmoc-O-Trt-hydroxy amino acids for solid-phase synthesis of    peptides. Tetrahedron Lett. 32(4):471-474.-   6. Barlos, K., P. Mamos, D. Papaioannou, S. Patrianakou, C. Sanida,    and W. Schaefer. 1987. Application of the Trt and Fmoc groups for    the protection of polyfunctional a-amino acid. Liebigs Ann. Chem.    12:1025-1030.-   7. Bieniariz, C., M. Husain, G. Barnes, C. A. King, and C. J.    Welch. 1996. Extended length heterofunctional coupling agents for    protein conjugations. Bioconjugate Chem. 7:88-95.-   8. Briggs, M. S. J., I. Bruce, J. N. Miller, C. J. Moody, A. C.    Simmonds, and E. Swann. 1997. Synthesis of functionalized    fluorescent dyes and their coupling to amines and amino acids. J.    Chem. Soc. Perkin Trans. 1. 7:1051-1058.-   9. Campbell, D. A. 1992. The synthesis of phosphonate esters; an    extension of the Mitsunobu reaction. J. Org. Chem. 57(23):6331-6335.-   10. Campbell, D. A., and J. C. Bermak. 1994. Phosphonate Ester    Synthesis Using a Modified Mitsunobu Condensation. J. Org. Chem.    59(3):658-660.-   11. Carlsson, J., H. Drevin, and R. Axen. 1978. Protein thiolation    and reversible protein-protein conjugation.    N-succinimidyl-3-(2-pyridyldithio)propionate, a new    heterobifunctional reagent. Biochem. J. 173:723-737.-   12. Carpino, L. A. 1993. 1-Hydroxy-7-azabenzotriazole. An efficient    peptide coupling additive. J. Am. Chem. Soc. 115(10):4397-4398.-   13. Carpino, L. A. 1987. The 9-fluorenylmethyloxycarbonyl family of    base-sensitive amino-protecting groups. Acc. Chem. Res.    20(11):401-407.-   14. Carpino, L. A., and G. Y. Han. 1972. 9-Fluorenylmethoxycarbonyl    amino-protecting group. J. Org. Chem. 37(22):3404-3409.-   15. Carpino, L. A., E. M. E. Mansour, C. H. Cheng, J. R.    Williams, R. MacDonald, J. Knapczyk, M. Carman, and A.    Lopusinski. 1983. Polystyrene-based deblocking-scavenging agents for    the 9-fluorenylmethyloxycarbonyl amino-protecting group. J. Org.    Chem. 48(5):661-665.-   16. Chamow, S. M., T. P. Kogan, D. H. Peers, R. A. Byrn, and A.    Askenaszi. 1992. Conjugation of soluble CD4 without loss of    biological activity via a novel carbohydrate-directed cross-linking    reagent. J. Biol. Chem. 267(22):15917-15922.-   17. De Bont, H. B. A., J. H. Van Boom, and R. M. J. Liskamp. 1990.    Automatic synthesis of phosphopeptides by phosphorylation on the    solid phase. Tetrahedron Lett. 31(17):2497-2500.-   18. Easton, C. J., and S. C. Peters. 1990. Synthesis of novel    crosslinked amino acid derivatives. Aust. J. Chem. 43(1):87-97.-   19. Evans, T. L., and M. M. Grade. 1986. Phase-transfer-controlled    selective oxidation of diaryl sulfides to diaryl sulfoxides using    potassium hydrogen persulfate. Synth. Commun. 16(10):1207-1216.-   20. Feldman, K., G. Haehner, N. D. Spencer, P. Harder, and M.    Grunze. 1999. Probing Resistance to Protein Adsorption of    Oligo(ethylene glycol)-Terminated Self-Assembled Monolayers by    Scanning Force Microscopy, p. 10134-10141, J. Am. Chem. Soc., vol.    121.-   21. Garegg, P. J., T. Regberg, J. Stawinski, and R.    Stroemberg. 1987. Studies on the oxidation of nucleoside hydrogen    phosphonates. Nucleosides Nucleotides. 6(1-2):429-432.-   22. Greenhalgh, R. P. 1992. Selective oxidation of phenyl sulfides    to sulfoxides or sulfones using Oxone and wet alumina. Synlett.    3:235-236.-   23. Harder, P., M. Grunze, R. Dahint, G. M. Whitesides, and P. E.    Laibinis. 1998. Molecular Conformation in Oligo(ethylene    glycol)-Terminated Self-Assembled Monolayers on Gold and Silver    Surfaces Determines Their Ability To Resist Protein Adsorption, p.    426-436, J. Phys. Chem. B, vol. 102.-   24. Hung, S. C., R. A. Mathies, and A. N. Glazer. 1998. Comparison    of Fluorescence Energy Transfer Primers with Different    Donor-Acceptor Dye Combinations. Anal. Biochem. 255(32).-   25. Jo, S., P. S. Engel, and A. G. Mikos. 2000. Synthesis of    poly(ethylene glycol)-tethered poly(propylene fumarate) and its    modification with GRGD peptide. Polymer. 41(21):7595-7604.-   26. Katritzky, A. R., B. Nowak-Wydra, and C. M. Marson. 1987. The    preparation of symmetrical secondary alkyl bromides. Chem. Scr.    27(3):477-478.-   27. Kenausis, G. L., J. Voeroes, D. L. Elbert, N. Huang, R.    Hofer, L. Ruiz-Taylor, M. Textor, J. A. Hubbell, and N. D.    Spencer. 2000. Poly(L-lysine)-g-Poly(ethylene glycol) Layers on    Metal Oxide Surfaces: Attachment Mechanism and Effects of Polymer    Architecture on Resistance to Protein Adsorption. J. Phys. Chem. B.    104(3298-3309).-   28. Kocienski, P. J., G. Cernigliaro, and G. Feldstein. 1977.    Pheromone synthesis. 4. A synthesis of (±)-methyl    n-tetradeca-trans-2,4,5-trienoate, an allenic ester produced by the    male dried bean beetle Acanthoscelides obtectus (Say). J. Org. Chem.    42(2):353-355.-   29. Konig, W., and R. Geiger. 1970. A new method for synthesis of    peptides: activation of the carboxyl group with    dicyclohexylcarbodiimide using 1-hydroxybenzotriazoles as additives.    Chem. Ber. 103(3):788-798.-   30. Kropp, P. J., G. W. Breton, J. D. Fields, J. C. Tung, and B. R.    Loomis. 2000. Surface-Mediated Reactions. 8. Oxidation of Sulfides    and Sulfoxides with tert-Butyl Hydroperoxide and OXONE. J. Am. Chem.    Soc. 122(18):4280-4285.-   31. Lemaitre, M., B. Bayard, and B. Lebleu. 1987. Specific antiviral    activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide    sequence complementary to vesicular stomatitis virus N protein mRNA    initiation site. Proc. Natl. Acad. Sci. U.S.A. 84(3):648-652.-   32. Lewis, M. R., and J. E. Shively. 1998.    Maleimidocysteineamido-DOTA derivatives new reagents for radiometal    chelate conjugation to antibody sulfylhydryl groups undergo    pH-dependent clevage reactions. Bioconj. Chem. 9:72-86.-   33. Lindh, I., and J. Stawinski. 1989. A general method for the    synthesis of glycerophospholipids and their analogs via    H-phosphonate intermediate. J. Org. Chem. 54(6): 1338-1342.-   34. Morpurgo, M., E. A. Bayer, and M. Wilchek. 1999.    N-hydroxysuccinimide carbonates and carbamates are useful reactive    reagents for coupling ligands to lysines on proteins. J. Biochem.    Biophys. Methods. 38(1):17-28.-   35. O'Shannessy, D. J., and R. H. Quarles. 1985. Specific    conjugation reactions of the oligosaccharide moieties of    immunoglobins. J. Applied Biochem. 7(347-355).-   36. Peng, L., G. J. Calton, and J. W. Burnett. 1987. Effect of    borohydride reduction on antibodies. Appl. Biochem. Biotechnol.    14:91-99.-   37. Perich, J. W., P. F. Alewood, and R. B. Johns. 1986.    Solution-phase synthesis of an O-phosphoseryl-containing peptide    using phenyl phosphorotriester protection, p. 1373-6, Tetrahedron    Lett., vol. 27.-   38. Perich, J. W., and E. C. Reynolds. 1991. Fmoc/solid-phase    synthesis of Tyr(P)-containing peptides through t-butyl phosphate    protection, p. 572-5, Int. J. Pept. Protein Res., vol. 37.-   39. Perich, J. W., R. M. Valerio, and R. B. Johns. 1986. Solid-phase    synthesis of an O-phosphoseryl-containing peptide using phenyl    phosphorotriester protection, p. 1377-80, Tetrahedron Lett., vol.    27.-   40. Saady, M., L. Lebeau, and C. Mioskowski. 1995. Direct    esterification of phosphonic acid salts using the Mitsunobu    reaction. Synlett. 6:643-644.-   41. Seigel, R. R., P. Harder, R. Dahint, M. Grunze, F. Josse, M.    Mrksich, and G. M. Whitesides. 1997. Online Detection of Nonspecific    Protein Adsorption at Artificial Surfaces, p. 3321-3328, Anal.    Chem., vol. 69.-   42. Song, X., and B. I. Swanson. 1999. Direct, Ultrasensitive, and    Selective Optical Detection of Protein Toxins Using Multivalent    Interactions, p. 2097-2107, Anal. Chem., vol. 71.-   43. Staerkaer, G., M. H. Jakobsen, C. E. Olsen, and A. Holm. 1991.    Solid phase peptide synthesis of selectively phosphorylated    peptides. Tetrahedron Lett. 32(39):5389-5392.-   44. Trost, B. M., and R. Braslau. 1988. Tetra-n-butylammonium oxone.    Oxidations under anhydrous conditions. J. Org. Chem. 53(3):532-537.-   45. Uto, I., T. Ishimatsu, H. Hirayama, S. Ueda, J. Tsuruta, and T.    Kambara. 1991. Determination of urinary Tamm-Horsfall protein by    ELISA using a maleimide method for enzyme-antibody conjugation. J.    Immunol. Meth. 138:87-94.-   46. Valerio, R. M., A. M. Bray, N. J. Maeji, P. O. Morgan, and J. W.    Perich. 1995. Preparation of O-phosphotyrosine-containing peptides    by Fmoc solid-phase synthesis: evaluation of several    Fmoc-Tyr(PO3R2)-OH derivatives. Lett. Pept. Sci. 2(1):33-40.-   47. van der Horst, G. T. J., G. M. S. Mancini, R. Brossmer, U. Rose,    and F. W. Verheijen. 1990. Photoaffinity labeling of a bacterial    sialidase with an aryl azide derivative of sialic acid. J. Biol.    Chem. 265(19):10801-10804.-   48. Verny, M., and C. Nicolas. 1988. [3H]-Labeling of hydroxyethyl    groups—synthesis of S-(2-hydroxy-[2-3H]-ethyl)glutathione and of    [3H]melphalan. J. Labelled Compd. Radiopharm. 25(9):949-955.-   49. Wagner, A., M. P. Heitz, and C. Mioskowski. 1989. Direct    conversion of tetrahydropyranylated alcohols to the corresponding    bromides. Tetrahedron Lett. 30(5):557-558.-   50. Wakamiya, T., K. Saruta, S. Kusumoto, S. Aimoto, K.    Yoshizawa-Kumagaye, and K. Nakajima. 1993. Synthetic study of    phosphopeptides by solid-phase method. Pept. Chem. 31:17-20.-   51. Wakamiya, T., K. Saruta, J. Yasuoka, and S. Kusumoto. 1994. An    efficient procedure for solid-phase synthesis of phosphopeptides by    the Fmoc strategy. Chem. Lett. 6:1099-1102.-   52. Ward, R. S., D. W. Roberts, and R. L. Diaper. 2000. Selective    oxidation of diaryl sulfides to sulfones with potassium hydrogen    persulfate. Sulfur Lett. 23(3):139-144.-   53. White, P. D. 2000. Fmoc Solid Phase Peptide Synthesis: A    Practical Approach. Oxford University Press, Oxford, UK.-   54. Xu, Q., E. A. Ottinger, N. A. Sole, and G. Barany. 1997.    Detection and minimization of H-phosphonate side reaction during    phosphopeptide synthesis by a post-assembly global phosphorylation    strategy. Lett. Pept. Sci. 3(6):333-342.-   55. Zalipsky, S. 1995. Chemistry of polyethylene glycol conjugates    with biologically active molecules. Adv. Drug Delivery Rev.    16(2,3):157-182.-   56. Zalipsky, S. 1995. Functionalized Poly(ethylene glycols) for    Preparation of Biologically Relevant Conjugates. Bioconjugate Chem.    6(2):150-165.-   57. Zalipsky, S., and J. M. Harris. 1997. Introduction to chemistry    and biological applications of poly(ethylene glycol). ACS Symp. Ser.    (Poly(ethylene glycol). 680:1-13.

Each of the above references is hereby incorporated by reference.

Although the present invention has been described with reference tospecific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

1. A sensor for detection of tetrameric multivalent neuraminidase withina sample, where a positive detection indicates presence of a targetvirus containing tetrameric multivalent neuraminidase within saidsample, said sensor comprising: a surface; at least two differentrecognition molecules each bound through a spacer group of a definedlength to an individual trifunctional linker moiety situated movably atsaid surface through an alkyl chain, said recognition molecules having abinding affinity with tetrameric multivalent neuraminidase, wherein atleast one individual trifunctional linker moiety includes a singlefluorescence donor label thereon for generating a fluorescent signalupon a predetermined binding event and at least one individualtrifunctional linker moiety includes a single fluorescence acceptorlabel thereon; and, a means for measuring for change in fluorescentproperties in response to binding between said two different recognitionmolecules and said tetrameric neuraminidase where a change influorescence indicates presence of said target virus.
 2. A method ofdetecting tetrameric multivalent neuraminidase within a sample, where apositive detection indicates presence of a target virus containingtetrameric multivalent neuraminidase within said sample, said methodcomprising: contacting a sample with a sensor including a surface, atleast two recognition molecules each bound through a spacer group of adefined length to an individual trifunctional linker moiety situatedmovably at said surface through an alkyl chain, said recognitionmolecules having a binding affinity with tetrameric multivalentneuraminidase wherein at least one individual trifunctional linkermoiety includes a single fluorescence donor label thereon for generatinga fluorescent signal upon a predetermined binding event and at least oneindividual trifunctional linker moiety includes a single fluorescenceacceptor label thereon; and measuring a change in fluorescent propertiesin response to binding between said two different recognition moleculesand said tetrameric neuraminidase.
 3. The sensor of claim 1 wherein saidvirus is influenza.
 4. The method of claim 2 wherein said virus isinfluenza.